Cellulases or cellulolytic enzymes are enzymes involved in hydrolysis of cellulose. In the hydrolysis of native cellulose, it is known that there are three major types of cellulase enzymes involved, namely cellobiohydrolase (1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.91), endo-beta-1,4-glucanase (endo-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.4) and beta-glucosidase (EC 3.2.1.21).
Especially the endoglucanases (EC No. 3.2.1.4) constitute an interesting group of hydrolases for the mentioned industrial uses. Endoglucanases catalyses endo hydrolysis of 1,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxy methyl cellulose and hydroxy ethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such as cereal beta-D-glucans or xyloglucans and other plant material containing cellulosic parts. The authorized name is endo-1,4-beta-D-glucan 4-glucano hydrolase, but the abbreviated term endoglucanase is used in the present specification. Reference can be made to T.-M. Enveri, "Microbial Cellulases" in W. M. Fogarty, Microbial Enzymes and Biotechnology, Applied Science Publishers, p. 183-224 (1983); Methods in Enzymology, (1988) Vol. 160, p. 200-391 (edited by Wood, W. A. and Kellogg, S. T.); Beguin, P., "Molecular Biology of Cellulose Degradation", Annu. Rev. Microbiol. (1990), Vol. 44, pp. 219-248; Beguin, P. and Aubert, J-P., "The biological degradation of cellulose", FEMS Microbiology Reviews 13 (1994) p.25-58; Henrissat, B., "Cellulases and their interaction with cellulose", Cellulose (1994), Vol. 1, pp. 169-196.
Cellulases are synthesized by a large number of microorganisms which include fungi, actinomycetes, myxobacteria and true bacteria but also by plants. Especially endoglucanases of a wide variety of specificities have been identified. Many bacterial endoglucanases have been described (Henrissat, B. and Bairoch, A. (1993) Biochem J. 293:781-788; Gilbert, H. J. and Hazlewood, G. P. (1993) J. Gen. Microbiol. 139:187-194).
The Clostridia subdivision is a very diverse group of anaerobic bacteria comprising physiologically very different genera. Previously, Clostridium was considered one genus including all endo spore-forming anaerobic bacteria. However the introduction of molecular taxonomic tools such as 16S rDNA sequencing have revealed that the group is heterogenous to a level far above genus. Moreover, various genera of non spore-forming anaerobes were classified within Clostridia, that turned out as a superior taxonomic group, a subdivision. This is in accordance to the highly diversified habitats for these organisms. The subdivision Clostridia, for example, comprises species with optimal growth temperature of a very broad range.
The genus Dictyoglomus comprises extreme thermophilic anaerobic bacteria phylogenetically situated within the Clostridia subdivision. Dictyoglomus spp. are among the most thermophilic organisms within the subdivision Clostridia. In the 16S rDNA phylogenetic tree Dictyoglomus occur with other thermophilic genera such as Thermoanaerobacter, Thermoanaerobacterium and Syntrophomonas as closest relatives. However Dictyoglomus form a deep branch confirming that Dictyoglomus indeed should be considered as a separate genus.
Dictyoglomus sp. strain B1 was isolated from a sludge and pulp sample from a pulpmass cooling tank, i.e. from a manmade thermophilic environment. A xylanase of this organism have been described with respect to temperature optimum (around 90.degree. C.). The xylanase production of this strain have been subjected to studies for fermentation optimization. The presence of endoglucanase from species of the genus Dictyoglomus was never reported. Within the phylum clostridia cellulases have been described from several species of which a few are thermophilic. In few cases the thermostability of endoglucanases have been determined, one of the most studied species is Clostridium thermocellum which have proven stable up to 80.degree. C. Thermoanaerobacter cellulyticus produces at least two endoglucanases with stability up to 80.degree. C.
Reference can be made to: Hudson et al. (1991) The cellulase activity of an extreme thermophile, Appl. Microbiol. Biotechnol. 35:270-273; Mathrani and Ahring (1991) Isolation and characterization of strictly xylan-degrading Dictyoglomus from man-made thermophilic environment, Arch. Microbiol. 157:13-17; Adamsen et al. (1995) Optimization of extracellular xylanase production by Dictyoglomus sp B1 in continuous-culture, Appl. Microbiol. Biotechnol. 44:327-332; Maidak et al. (1994) The Ribosomal Database Project, Nuc. Acids Res. 22:3485-3487; Honda et al. (1987) Cloning and expression in E.coli of a Thermoanaerobacter cellulyticus gene encoding for heatstable beta-glucanase, Appl. Microbiol. Biotechnol. 25:480-483; Honda et al. (1988) Isolation of a new cellulase gene from a thermophilic anaerobe and its expression in E. coli, Appl. Microbiol. Biotechnol. 29:264-268.
A very important industrial use of cellulolytic enzymes is the use for treatment of cellulosic textile or fabric, e.g. as ingredients in detergent compositions or fabric softener compositions, for bio-polishing of new fabric (garment finishing), and for obtaining a "stone-washed" look of cellulose-containing fabric, especially denim, and several methods for such treatment have been suggested, e.g. in GB-A-1 368 599, EP-A-0 307 564 and EP-A-0 435 876, WO 91/17243, WO 91/10732, WO 91/17244, PCT/DK95/000108 and PCT/DK95/00132. Another important industrial use of cellulytic enzymes is the use for treatment of paper pulp, e.g. for improving the drainage or for deinking of recycled paper.
It is also known that cellulases may or may not have a cellulose binding domain (a CBD). The CBD enhances the binding of the enzyme to a cellulose-containing fiber and increases the efficacy of the catalytic active part of the enzyme.
There is a need for providing economically feasible cellulase enzyme preparations which may be used for applications where cellulase, preferably an endoglucanase, activity at high temperatures is desirable.
The object of the present invention is to provide novel enzyme compositions or recombinant enzymes having substantial cellulolytic activity at high temperature conditions and improved performance in industrial applications, e.g. in paper pulp processing, textile treatment, laundry processes, extraction processes or in animal feed.